摘要
目的：机体内前炎症递质肿瘤坏死因子α、白细胞介素6与抗炎症递质白细胞介素10之间的平衡出现严重紊乱，可导致内毒素休克及多脏器功能衰竭。实验拟进一步探讨含大鼠白细胞介素10基因重组复制缺陷型腺病毒体外转染骨髓基质细胞后，内毒素休克动物模型导入的白细胞介素10基因表达及其对机体肿瘤坏死因子α、白细胞介素6水平的调节。方法：实验于2006—05／2007-10在中山大学附属第一医院外科实验室完成。①材料：清洁级SD雌鼠100只，选取10只用于骨髓基质细胞的分离培养，剩余90只随机数字表法分为细胞转染组、模型组、正常组，30只/组，实验过程中对动物的处置符合动物伦理学标准。含大鼠白细胞介素10基因的重组复制缺陷型腺病毒由中山大学附属第一医院烧伤外科朱家源教授惠赠。内毒素Escherichia coli 0111：B4为美国Sigma公司产品。②实验方法：将含大鼠白细胞介素10基因的重组复制缺陷型腺病毒复苏后，加入到贴壁生长达80％的第3代骨髓基质细胞中，将转染后细胞、未转染细胞按5×10^5／孔接种，采用ELISA法和RT-PCR法检测体外白细胞介素10的含量变化。细胞转染组与模型组大鼠均经左侧股静脉注射内毒素5mg／kg建立休克模型，10min后细胞转染组在同侧股静脉注射转染后培养12h的5×10^8L^-1骨髓基质细胞0.5mL，模型组与正常组均注射未转染的5×10^8L^-1骨髓基质细胞0．5mL，分别于0，3，6，12，24，36，48h采用ELISA法检测体内各组大鼠外周血清白细胞介素10、白细胞介素6和肿瘤坏死因子α水平，并记录以上各时相点大鼠存活数。细胞回输48h后，组织病理学检测大鼠肝、肺、肾的变化。结果：①体外白细胞介素10的表达：体外转染后的骨髓基质细胞培养上清中可检测到白细胞介素10的表达，表达高峰位于转染后36h，于560bp处显示凝胶电泳条带；未经转染的骨髓基质细胞不表达白细胞介素10。②体内白细胞介素10、肿瘤坏死因子α和白细胞介素6的表达：与模型组比较，转染后3，6，12，24，36，48h细胞转染组外周血清白细胞介素10水平均显著上升（t=-15．51～30．98，P均〈0．05），肿瘤坏死因子α和白细胞介素6水平均显著下降0=-14、78～36．75，P均〈0．05；t=-12.49～38．14，P均〈0．05）。③大鼠存活情况：细胞回输后48h，细胞转染组大鼠存活率为50％，明显高于模型组20％（x^2=5．93，P〈0．05）。④组织病理学观察：模型组肝细胞肿胀、空泡样变性、汇管区大量炎性细胞浸润，中央静脉内有血栓形成，可见点片状肝窦结构被破坏；肺呈间质性肺炎改变，较多单核和中性粒细胞浸润，小血管壁内充血，局部可见坏死小灶；肾间质充血，肾小球体积增大，近曲小管上皮细胞肿胀、玻璃样变。细胞转染组肝、肺、肾损伤均明显减轻。结论：进行体外转染的骨髓基质细胞回输后，其所携带的白细胞介素10基因可实现体内外的高水平表达，对内毒素休克时肿瘤坏死因子α和白细胞介素6水平起到预期的下调作用，减轻了机体的炎症反应程度，并有效保护肝、肺、肾等主要脏器。
AIM： It can cause endotoxin shock and multiple organ dysfunctions when the balance between anterior inflammation transmitter tumor necrosis factor （TNF）- α, intedeukin （IL）-6 and anti-inflammation transmitter IL-10 has become serious disorder in the body. This study aimed to investigate the expression of IL-10 gene which is introduced into animal models of endotoxin shock by bone marrow stromal cells transfected in vitro with replication-deficient rat IL-10 recombinant adenovirus, and study the changes in TNF- α and IL-6 levels in the models.
METHODS： The experiment was performed at the laboratory of Department of General Surgery, First Affiliated Hospital of Sun Yat-sen University from May 2006 to October 2007. Ten rats of 100 clean female Sprague Dawley rats were selected for the isolation and culture of bone marrow stromal cells, and the remainings were divided into a cell transfection group, a model group and a normal group by random digits table method, with 30 rats in each group. The disposal to animals during the experiment was accorded with animal ethical standards. Replication-deficient rat IL-10 recombinant adenovirus was provided by professor Zhu from Department of Burn of First Affiliated Hospital of Sun Yat-sen University. Endotoxin Escherichia coli 0111： B4 （Sigma, USA） was used in the study. Replication-deficient rat IL-10 recombinant adenoviruses after recovery were added to the bone marrow stromal cells of the 3^rd generation, which had been adhered to the flask more than eighty percent, The cells transfected and untransfected were all inoculated according to 5× 10^5 per hole. The changes in IL-10 levels were detected by enzyme-labeled immunosorbent assay （ELISA） and reverse transcription polymerase chain reaction （RT-PCR） methods, The rat models of shock were established in the cell transfection group and model group by injecting 5 mg/kg endotoxin into left femoral vein. Ten minutes later, rats in the cell transfection group were injected with 0.5 mL of bone marrow stromal cells （5 × 10^8 L^-1） into the left femoral vein which had been ransfected and cultured for 12 hours. Rats in the model group and normal group were all injected with 0.5 mL of bone marrow stromal cells （5× 10^8 L^-1） into the left femoral vein which had not been transfected. The serum IL-10, TNF-α and IL-6 levels of rats in each group were measured by ELISA at 0, 3, 6, 12, 24, 36 and 48 hours, and the survival rats at the same time point were recorded. After 48-hours reinfusion, histopathological changes in liver, lung and kidney of the rats were tested. RESULTS： IL-10 expression could be checked in culture supernatant of bone marrow stromal cells transfected in vitro. The peak expression located at 36 hours after transfection. A band of 560 bp showed in agar gel electrophoresis. The bone marrow stromal cells, which had not been transfected, did not express IL-10. Compared to the model group, the serum IL-10 levels in the cell transfection group increased significantly at 3, 6, 12, 24, 36 and 48 hours after transfection （t = 15.51-30.98, P 〈 0.05 ） . Serum TNF- α and IL-6 levels in the cell transfection group decreased significantly at the same time（t=14.78-36.75, P〈0.05; t=12.49-38.14, P〈0.05）. After 48-hours reinfusion, the survival rate in the cell transfection group was （50%） significantly higher than in the model group （20%） （ x^2=5.93, P 〈 0.05）. The hepatocyte in the model group showed cell swelling and bubble-like degeneration, inflammatory cells infiltrated in portal area, thrombus formed in central vein, point and flake of liver sinus were destroyed. The lung showed interstitial pneumonia, and there were many monocytes and mononclear cells infiltration, hyperemia in wall of small vessels, and visible local small necrosis focus. Renal interstitium was hyperemic, the glomerular volume became bigger, and renal tubular epithelial cells showed swelling and glassy degeneration. The damages of liver, lung and kidney in the cell transfection group were not serious like those in the model group. CONCLUSION： After reinfusing with bone marrow stromal cells, which had been transfected in vitro, IL-10 gene carried by bone marrow stromal cells can highly express IL-10 inside or outside the body, and play a decreasing effect on TNF- α and IL-6 levels, when the body is facing endotoxin shock. This lightens the severity of acute inflammatory response, and effectively protects main organs such as lever, lung and kidney.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第21期4054-4059,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
深圳市科技和信息局科研基金(JH200505300509A)
深圳市宝安区科学技术局科研基金(2005104)~~
